Brain-targeted delivery of a leucine-enkephalin analogue by retrometabolic design

Article abstract of DOI:10.1021/jm960356e

A brain-targeted chemical delivery system (CDS) based on retrometabolic drug design was applied to a Leu-enkephalin analogue, Tyr-D-Ala-Gly-Phe-D- Leu (DADLE). The molecular architecture of the peptide CDS disguises its peptide nature from neuropeptide-de

Full text of DOI:10.1021/jm960356e

J . Med. Chem. 1996, 39, 4775-4782
4775
Br a in -Ta r geted Deliver y of a Leu cin e-en k ep h a lin An a logu e by Retr om eta bolic
Design †
Katalin Prokai-Tatrai, Laszlo Prokai, and Nicholas Bodor*
Center for Drug Discovery, College of Pharmacy, University of Florida, Gainesville, Florida 32610-0497
Received May 15, 1996X
A brain-targeted chemical delivery system (CDS) based on retrometabolic drug design was
applied to a Leu-enkephalin analogue, Tyr-D-Ala-Gly-Phe-D-Leu (DADLE). The molecular
architecture of the peptide CDS disguises its peptide nature from neuropeptide-degrading
enzymes and provides lipophilic, bioreversible functions for the penetration through the blood-
brain barrier. These functions were provided by a targetor, a 1,4-dihydrotrigonellyl group, on
the N-terminus and a bulky, lipophilic ester group on the C-terminus. A spacer amino acid
residue was also inserted between the targetor and the parent peptide to assure the release of
DADLE by specific enzymes. Four CDSs were synthesized by segment-coupling method that
proved to be superior to sequential elongation in obtaining this type of peptide conjugates.
Intravenous injection of the compounds produced a significant and long-lasting response in
rats monitored by the tail-flick latency measurements. CDSs having the bulkier cholesteryl
group showed a better efficacy than those having the smaller 1-adamantaneethyl ester. The
spacer was the most important factor to manipulate the rate of DADLE release and, thus, the
pharmacological activity; proline as a spacer produced more potent analgesia than alanine.
The antinociceptive effect of the CDSs was naloxone-reversible and methylnaloxonium-
irreversible, confirming that central opiate receptors were solely responsible for mediating
analgesia induced by the peptide CDS that delivered, retained, and then released the peptide
in the brain.
In tr od u ction
brain barrier (BBB). The BBB, which acts as a selective
partition between the CNS and peripheral nervous
system, is the major obstacle for delivery of centrally
active peptides to the brain.10 Peptides may also be
recognized by neuropeptide-degrading enzymes ex-
pressed in the BBB, even when their lipophilicity allows
their transport through the BBB.11 Recently, an en-
kephalin analogue12,13 and a centrally active thyrotro-
pin-releasing hormone14 have been successfully trans-
ported into the CNS by a brain-targeting chemical
delivery based on retrometabolic drug design ap-
proach.15,16 A brain-targeting peptide chemical delivery
system is defined as a biologically inert molecule which
requires several steps in its conversion to the biologi-
cally active peptide and ensures drug differential de-
livery to the CNS, the site of action. In this approach,
a redox “targetor” (1,4-dihydrotrigonellyl) moiety is
attached to the N-terminal amino acid of the peptide
chain via a spacer amino acid, and a bulky, lipophilic
moiety is anchored to the C-terminus via an ester bond.
The dihydropyridine-type targetor has definite roles.
This group enhances BBB penetration and, most im-
portantly, can be converted by enzymatic oxidation to
a water-soluble, lipid-insoluble pyridinium salt that
cannot leave the brain but can easily be eliminated from
the peripheral circulation. A spacer amino acid residue
separates the targetor from the biologically active
peptide to enhance its release by specific enzymes from
the inactive conjugate. This cleavage should be favored
over proteolytic degradations induced by other pepti-
dases. Attachment of the 1,4-dihydrotrigonellyl to the
N-terminus alone would not give significant increase in
lipid solubility to a peptide and would only protect
against aminopeptidases such as aminopeptidase N (EC
3.4.11.2). Therefore, a bulky ester on the C-terminus
is applied, which not only protects this part of the
Enkephalins are naturally occurring linear pentapep-
tides that bind to opioid receptors and have the sequence
Tyr-Gly-Gly-Phe-Leu/Met ([Leu]enkephalin or [Met]-
enkephalin).1 The action of these endogenous peptides
on pain perception, addictive states, and psychiatric
disorders have been thoroughly investigated.2 However,
analgesia is their best-known central effect.3,4 The
metabolic stability of enkephalins is poor. Studies
suggest that at least four different types of enkephalin-
metabolizing enzymes contribute to their rapid deacti-
vation. The enkephalins are predominantly cleaved by
aminopeptidases5 at the N-terminal tyrosine. En-
kephalinase and angiotensin-converting enzymes6 cleave
the Gly3-Phe4 bond, and a dipeptidyl aminopeptidase7
cleaves the Gly2-Gly3 bond. There have been numerous
attempts to modify the original structure to improve
metabolic stability and receptor binding (affinity, se-
lectivity).8 One approach to stabilize the peptide back-
bone against proteases incorporates unnatural D-amino
acids to the peptide chain, such as replacements of Gly2
with D-Ala and Leu with D-Leu.9 Metabolic stability
alone does not, however, warrant access of the peptide
analogues to the central nervous system (CNS) upon
systemic administration.
Many peptides also are potential neuropharmaceuti-
cals. Most of them are water soluble and cannot
penetrate through the lipoidal bilayer of the blood-
† Abbreviations: DCC, 1,3-dicyclohexylcarbodiimide; DMAP, 4-(di-
methylamino)pyridine; HOBt, 1-hydroxybenzotriazole; PyBOP, ben-
zotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate; DCM,
dichloromethane; DMF, N,N-dimethylformamide; Fmoc, fluorenyl-
methyloxycarbonyl; Boc, tert-butyloxycarbonyl; EtOAc, ethyl acetate;
MeOH, methanol; TFA, trifluoroacetic acid; NMP, N-methylpyrroli-
dinone; DIEA, N,N-diisopropylethylamine; TEA, triethylamine; Nic,
t
nicotinoyl; Bu, tert-butyl; Su, N-hydroxysuccinyl.
X Abstract published in Advance ACS Abstracts, November 1, 1996.
S0022-2623(96)00356-1 CCC: $12.00 © 1996 American Chemical Society

4776 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Prokai-Tatrai et al.
Sch em e 1. Brain Targeting of DADLE by
Retrometabolic Design
cholesteryl (Cho) or 1-adamantaneethyl (Ada), anchored
to the COOH-terminus via an ester bond provides
protection of this part of the molecule. A 1,4-dihydro-
trigonellyl targetor (T) on the N-terminus enhances
BBB transport and, most importantly, after reaching
the CNS keeps the peptide conjugate in the brain due
to the enzymatic oxidation to a lipid-insoluble ionic salt
(T+, trigonellyl). The mechanism of this oxidation has
been extensively studied, and it has been suggested to
be analogous to the oxidation of NAD(P)H, a coenzyme
associated with numerous oxidoreductases and cellular
respiration.18 Attachment of the targetor directly to the
amino-terminal residue may not afford the release of
the target peptide at a desired rate because of the low
amidase activity of the brain tissue.19 Therefore, a
spacer (S) function is also introduced into the peptide
chain for separation of the enkephalin moiety from the
targetor to promote the cleavage that yields DADLE.
The selection of alanine and proline as a spacer is based
on the suggested involvement of alanyl and prolyl
aminodipeptidyl peptidases in the enkephalinergic trans-
mission in the brain.20,21 These enzymes have specific
cleaving site in the peptide sequence. We assumed that
trigonelline (synthetic precursor and oxidative metabo-
lite of T) represents an amino acid residue for these
dipeptidyl peptidases because it is attached to the spacer
by a peptide-like bond and has a permanent positive
charge like an N-terminal amino acid. Furthermore,
pyridinium salt-type amino acids can replace natural
ones in various peptides such as LHRH.22
After passive transport through the BBB the targetor
undergoes an enzymatically mediated oxidation in the
CNS to a lipid-insoluble trigonellyl (T+) salt that is
retained (“locked-in”) in the brain. Thus, unlike a
lipophilic peptide prodrug,23 T+-S-DADLE-OLpf cannot
leave the brain. It has been shown13,14 that the trapped
peptide conjugate undergoes enzymatic cleavages re-
leasing the parent peptide in a slow and sustained
manner. The lipophilic ester group is cleaved by lipases
and/or esterases after or during penetration through the
BBB.
peptide from peptidases but also ensures the necessary
lipophilicity for passive transport of the peptide conju-
gate through the BBB into the CNS. Simple alkyl esters
do not provide these functions.12 The ester bond is
stable chemically but labile enzymatically. Sequential
metabolism (involving oxidoreductases, lipases/esteras-
es, dipeptidyl peptidases) in the brain affords the
retention (“lock-in”) and the subsequent release of the
biologically active peptide.
In an apparent effort to improve and optimize this
strategy, the objectives of our study were to develop an
effective synthesis strategy for obtaining peptide chemi-
cal delivery systems (CDSs) and to determine the
potential contribution of the spacer and the C-terminal
ester, the two functions that may be adjusted to achieve
optimal brain targeting of Tyr-D-Ala-Gly-Phe-D-Leu
(DADLE), to the efficacy of CDSs to induce CNS-
mediated analgesia.
Syn th esis. The CDSs of DADLE for brain-targeted
delivery were obtained by two routes; both applied the
segment-coupling peptide synthesis strategy. Ini-
tially,13,15 peptide CDSs were prepared by DCC/HOBt
coupling via a stepwise elongation of the peptide chain
in the C to N direction starting with the lipophilic
cholesteryl ester of the C-terminal residue. This cou-
pling method was time-consuming and inconvenient
because frequent purification was required. We have
improved this initial procedure according to Scheme 2
(method A) by combining the step by step elongation of
the peptide chain with segment condensation and using
active esters instead of DCC activation. Peptides esters
having a Cho ester group on the C-terminus and a Nic
residue on the N-terminus (8a ,b) were obtained after a
dipeptide-pentapeptide (2 + 5) coupling. The pen-
tapeptide represented the lipophilic ester of the en-
kephalin DADLE-OLpf (7, an enkephalin prodrug). It
was prepared by stepwise elongation of the peptide
chain with N-hydroxysuccinimide esters of the Boc-
protected amino acids. The first step in the synthesis
of 7 involved the preparation of the lipophilic ester of
D-leucine (3) that was easily achieved by either DCC/
DMAP or PyBOP/DIEA24 activation. Then, the Boc
protecting group was removed in a customary manner,
Resu lts
Design . A chemical delivery system consistent with
the activation mode of retrometabolic concept was
developed for brain-targeting delivery of enkephalin
analogues.15,16 The CDS should be sufficiently lipophilic
for allowing brain uptake and should undergo an
enzymatic conversion to promote retention within the
CNS. In this design, shown in Scheme 1, a synthetic
leucine-enkephalin analogue, DADLE, a well-known
opioid agonist,17 is surrounded by functional units that
protect the peptide from being recognized by peptide-
degrading enzymes and provide biolabile and lipophilic
functions for the penetration through the BBB by
passive transport. A bulky, lipophilic group (Lpf),

CDS of a Leu-enkephalin Analogue
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24 4777
Sch em e 2. Synthesis of 10a ,b by Method Aa
The dipeptide segment in this procedure was the
pentafluorophenyl ester of the N-terminal dipeptide,
Nic-S-OPfp (2a ,b), prepared from Nic-S-OH (1a ,b) and
pentafluorophenol with DCC.25 Compounds 1a ,b were
synthesized from Nic-Cl and Ala/Pro-OtBu followed by
in situ hydrolysis of the ester group with TFA/DCM (1:
1, v/v). The two fragments, 2a ,b and 7, were assembled
in DMF with excellent yield without any auxiliary
additives resulting in compounds 8a ,b. The reaction
was complete within 1 day using a 2-fold excess of 2a
or 2b. At this stage, we applied the only chromato-
graphic purification in this procedure for ensuring the
purity of the final products, the peptide CDSs. The
peptide esters 8a ,b were purified by column chroma-
tography on silica gel. Because they showed limited
solubility in the chromatographic solvents, they were
dissolved in chloroform/MEOH (6:4, v/v), evaporated on
silica gel, and put on the top of the column. Using
chloroform/MEOH (9:1, v/v) containing 0.1% TEA as an
eluent, pure products 8a ,b were obtained. After alky-
lation on the pyridine ring with methyl iodide, the
obtained pyridinium salts 9a ,b were reduced with
sodium dithionite26 to the corresponding peptide CDS
10a ,b.
For an alternative route (method B), we chose the
hexapeptide-single amino acid (6 + 1) segment coupling
and combined solid phase peptide synthesis (SPPS) with
solution phase synthesis. As Scheme 3 shows, the two
individual segments in this case were the lipophilic ester
of the C-terminal amino acid 11 and the residual
fragment of the peptide chain Nic-S-Tyr-D-Ala-Gly-Phe-
OH (12a ,b), respectively. This approach is illustrated
with the synthesis of CDSs containing Ada ester group
on the C-terminus (15a ,b). The hexapeptides were
prepared by SPPS using an automated Fmoc chemistry
protocol. The DCC-mediated coupling was accelerated
with HOBt. After cleavage from the resin, the crude
peptide compounds 13a ,b were identified by FAB-MS
and used without further purification. Esterification of
D-Leu was carried out the same way as in method A for
compound 3. The PyBOP/DIEA system was also se-
lected for the preparation of 13a ,b. The segment
coupling was complete (monitored by TLC and FAB-MS)
within 1 day using approximately 1.5-fold excess of
a
(i) PyBOP/DIEA; (ii) TFA/TEA. a : S ) Pro, b: S ) Ala.
and triethylamine was used for neutralization. Com-
pound 3 was elongated with a slight excess of the active
ester of the relevant Boc-protected amino acid. The
progress of the coupling reactions was monitored by
TLC and fast-atom bombardment mass spectrometry
(FAB-MS). The intermediate peptides obtained after
recrystallization were of sufficient purity (FAB-MS,
TLC) to be used directly for the next peptide bond
formation. Therefore, no attempts have been made to
further characterize and/or purify the pendant peptide
chain leading to 7.
Sch em e 3. Preparation of 15a ,b by Method Ba
a
(i) PyBOP/DIEA; (ii) TFA/TEA. a : S ) Pro, b: S ) Ala.

4778 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Prokai-Tatrai et al.
F igu r e 1. Analgesic activity of DADLE CDSs in rats, measured by the tail-flick latency, at 4.2 µmol/kg of body weight and
intravenous injection: (a) compound 10a (Lpf ) Cho, S ) Pro), (b) compound 10b (Lpf ) Cho, S ) Ala), (c) compound 15a (Lpf
) Ada, S ) Pro), and (d) compound 15b (Lpf ) Ada, S ) Ala). Data are represented as average ( SEM. Dashed lines represent
the control group (injection of vehicle).
12a ,b in a solvent mixture of DMF/dioxane. After
removing the volatiles, the crude products 13a ,b were
carefully purified by column chromatography the same
way as 8a ,b. Methylation of the pyridine ring (14a ,b)
and the subsequent reduction resulting in the CDSs
15a ,b followed the same routine we applied for method
A.
We have also investigated another approach for the
preparation of peptide CDSs, in which the direct esteri-
fication on the C-terminus of the entire heptapeptide
Nic-S-DADLE-OH was studied. However, the ester
bond formation either with cholesterol or with 1-ada-
mantaneethanol proved to be very inefficient and not
reproducible. Therefore we did not consider this route
for obtaining peptide CDS.
In Vitr o Sta bility a n d Recep tor Bin d in g. Stabil-
ity studies of the CDSs and their predicted biotrans-
formation products have been performed in phosphate
buffer, whole blood, and 20% (w/w) brain homogenate.
An experimental formulation that involved inclusion
complex of the CDS with a chemically modified cyclo-
dextrin has also been developed. The CDSs were
unstable, as expected, in biological fluid due to oxida-
tion. All the quaternary intermediates (T+-S-DADLE-
OLpf (9a ,b/14a ,b) and T+-S-DADLE-OH (16a ,b)) were
stable in the pH range 4-9; no decomposition occurred
over 6 h. The oxidized CDS 9 had half-lives of around
2-3 min in whole blood and around 35-40 min in brain
homogenate. The quaternary acids 16 were stable in
whole blood and had a half-life of 4.9 h for 16a , while
16b was more stable (t1/2 > 12 h). The sustained release
of DADLE was unequivocally shown by electrospray
ionization mass spectrometric analysis, as given in the
Supporting Information.
The receptor binding study (competition with [3H]-
diprenorphine, a µ- and δ-receptor agonist) confirmed
that 9 had very low (IC50 ≈ 6 µM) affinity, while we
measured weak binding (IC50 > 200 nM)27 to opioid
receptors for 16, as compared to DADLE (IC50 ) 36 nM).
Competitive binding that involved a µ-selective radio-
ligand ([3H]DAGO) also showed about 2 orders of
magnitude difference in affinity between DADLE and
16. Therefore, it can be concluded that only a “real”
chemical delivery of DADLE results in a profound
antinociceptive response.
An a lgesic Activity. The antinociceptive properties
of the CDSs of DADLE (10a ,b and 15a ,b) were evalu-
ated by tail-flick latency measurement upon iv admin-
istration (4.2 µmol/kg of body weight), similar to those
in the preliminary studies.12,13 We observed a statisti-
cally significant and prolonged (more than 5 h) increase
in the tail-flick latency for all of the tested CDSs, as
revealed in Figure 1d. Compounds having proline as a
spacer (10a and 15a ) reached their maximum analgesic
effect earlier (between 15 and 30 min after the admin-
istration of the drug) than those containing an alanine
spacer. The CDSs with the smaller, thus less lipophilic,
ester group on the C-terminus (15a ,b) showed lower
analgesic activity compared to those with Cho.
We selected compound 10a (S ) Pro, Lpf ) Cho), as
the most potent CDS among those tested, for further
study. Naloxone, an opiate antagonist,28 was injected
subcutaneously at a dose of 2.7 µmol/kg of body weight,
and the tail-flick latency was monitored. The analgesic

CDS of a Leu-enkephalin Analogue
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24 4779
the release of DADLE had weak affinity to opioid
receptors. Thus, the observed analgesia was mostly due
to the enkephalin analogue liberated at the site of
action. The selection of S and Lpf can greatly influence
the efficacy of the CDS. The lipophilicity/size of Lpf is
evidently a basic requirement for the successful delivery
of the peptide through the BBB. Compounds with Cho
on the C-terminus produced higher tail-flick latencies
compared to those having Ada. Consequently, the size
of Lpf per se has an important contribution to the
successful delivery of DADLE. (Ada should provide
enough lipid solubility to the peptide conjugate; the
calculated log P values are >2 for 15a ,b.)30 On the other
hand, the rate of the release of DADLE is most signifi-
cantly influenced by the spacer. For the proline spacer
containing CDSs, the maximum of the antinociceptive
effect was reached earlier than for those containing the
Ala spacer, independently of the size of Lpf, due to the
different affinity of Pro and Ala to the dipeptidyl
peptidases.20,21 The in vitro experiments with the
relevant biotransformed intermediates 16a ,b comple-
mented these observations. The measured t1/2 in 20%
brain homogenate is 3-4 times shorter for 16a (S ) Pro)
than for 16b (S ) Ala). By manipulating the spacer,
the analgesic efficacy of the CDS can, thus, be modified
and predicted according to the therapeutic objective.
Based on our experiments, for a potentially strong, fast-
acting analgesic, the molecular packaging should con-
tain S ) Pro and Lpf ) Cho functions. We have also
shown that the antinociceptive effect of DADLE brain
targeted by a CDS can completely be antagonized by
naloxone, an opioid antagonist that crosses the BBB
(Figure 2). This observation was complemented by an
experiment using a quaternary analogue of naloxone
that cannot cross the BBB and thus may interact only
with peripheral opioid receptors. Indeed, the naloxo-
nium salt did not reverse the analgesic effect of the
tested compound. These results provide evidence for the
exclusively centrally mediated analgesia induced by the
CDS due to its specific design that delivered, retained,
and then released DADLE in the brain.
F igu r e 2. Reversal of the analgesia of 10a (4.2 µmol/kg of
body weight, iv, dotted line) by naloxone (2.7 µmol/kg of body
weight, sc, solid line), but not by naloxonium (2.1 µmol/kg of
body weight, sc, dashed line), in rats.
effect was completely antagonized by naloxone (Figure
2), providing evidence for the centrally mediated anal-
gesia induced by the peptide CDS. This observation was
further confirmed by administering naloxone methyl
iodide, the quaternary derivative of naloxone that does
not cross the BBB, under the same experimental condi-
tions (Figure 2). However, the antinociceptive effect of
the CDS was not reversed by the peripherally acting
opioide antagonist.
Discu ssion
For the further refinement of an already successful
targeting of DADLE to the brain12,13 by a CDS approach,
we have evaluated the spacer (Ala or Pro) and the size/
lipophilicity of the ester group (Cho or Ada) for the
efficacy of the CDSs to induce CNS-mediated analgesia
upon iv administration. For these studies, it was
necessary to develop an effective synthesis strategy for
obtaining peptide CDSs. We have successfully applied
the segment-coupling strategy for a fast and straight-
forward synthesis of this type of peptide conjugate.
Both methods reported here (methods A and B) are also
equally effective and reproducible for large scale prepa-
ration without frequent, time-consuming purifications
of the pendant peptide chain, independently of the size
of Lpf. Even using a solely solution phase approach
involving active ester (method A), the side chain protec-
tion of tyrosine can be avoided,29 which further simpli-
fies the procedure and the purification of the key
compounds Nic-S-DADLE-OLpf. The purity of these
peptide esters with a Nic residue on the N-terminus
(8a ,b, and 13a ,b) is crucial for obtaining pure CDSs.
Although the remaining two subsequent reaction steps
(methylation and reduction) leading to the peptide CDS
are straightforward, the chromatographic purification
of their product, 9a ,b/14a ,b and 10a ,b/15a ,b, respec-
tively, is cumbersome and very inefficient. Using pure
precursors (Nic-S-DADLE-OLpf), however, the CDSs
with excellent purity can be obtained without trouble-
some purification.
In conclusion, the brain-targeted delivery of DADLE
can be refined and optimized according to the thera-
peutic objective by manipulating the lipophilic moiety
(Lpf), and the spacer (S) function that affects the rate
of peptide release in the CNS. Furthermore, due to the
rational design of the CDS, the induced analgesia is
solely centrally mediated.
Exp er im en ta l Section
In str u m en ts a n d Ma ter ia ls. All chemicals used were of
reagent or peptide synthesis grade. N-Hydroxysuccinimide
esters of Boc-protected amino acids were obtained from
Bachem Bioscience, Inc. (Philadelphia, PA). Solvents and
other chemicals were purchased from Fisher Scientific, Inc.
(Pittsburgh, PA). Fmoc-phenylalanine p-benzyl alcohol resin,
100-200 mesh, 0.78 mequiv/g on 1% DVB was purchased from
Chem-Impex Int. (Wood Dale, IL). Naloxone hydrochloride
and naloxone methiodide were obtained from Research Bio-
chemicals International (Natick, MA). Thin layer chromatog-
raphy was performed on silica gel-coated (E. Merck Kieselgel
60 F-254, 0.2-mm thickness) plates and developed with
chloroform/MEOH, 9:1, v/v. Spots were located either with
UV light or by treatment of the plate with HCl fumes followed
by heating and subsequent spraying with ninhydrin. Organic
solutions that had been previously extracted with aqueous
solution were dried over anhydrous Na2SO4. Column chro-
matographies were performed using Davisil grade 643 silica
The CDSs were tested for analgesic efficacy based on
tail-flick latency measurements in rats. All of the
compounds showed a profound, long-lasting analgesia
as illustrated in Figures 1 and 2. Our studies revealed
that the packaged peptide and the intermediates formed
during the sequential metabolism ultimately leading to
gel. Melting points were determined on
a Fisher-J ohns

4780 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Prokai-Tatrai et al.
apparatus and uncorrected. Elemental analyses were supplied
by Atlantic Microlabs, Inc. (Norcross, GA). FAB mass spectra
were recorded on a Kratos MS80RFA instrument (Kratos
Analyticals, Manchester, U.K.), xenon beam (8 keV energy),
by dissolving the sample in 3-nitrobenzyl alcohol matrix with
or without Na.31 UV spectra were recorded in methanol on a
Lambda 11 UV/vis Perkin Elmer (Perkin Elmer Anal. Inst.,
Norwalk, CT) spectrophotometer. A Synthor 2000, Peptide
International (Louisville, KY), instrument was used for SPPS.
RP-HPLC purification was performed on a system consisting
of a ThermoSeparation/SpectraPhysics (Fermont, CA) Spectra
Series P200 binary gradient pump, a Rheodyne (Cotati, CA)
TEA under ice cooling. The Boc-amino acid-OSu (15-20%
excess to the lipophilic ester) was added, the cooling was
removed, and the reaction mixture was stirred at room
temperature for 1-2 days (monitored by TLC and/or FAB-MS).
The solution was diluted with DCM and extracted with ice
cold 1 N HCl (2×), saturated NaHCO3 (2×), and brine (2×),
respectively. The organic layer was dried and evaporated in
vacuo. The crude peptide product was identified by FAB-MS
and used after recrystallization from chloroform/ether. The
yields (for crude peptides) were 70-80%.
Boc-DADLE-OCh o (7). An analytical sample was ob-
tained after column chromatographic purification using chlo-
roform/MeOH (95:5, v/v) containing 0.1% TEA as an eluent;
white solid; mp 121-122 °C dec; Rf ) 0.42; MS [M + Na]+
m/ z 1060. Anal. (C61H91N5O9‚1.5 H2O) C, H, N.
Meth od A: (2 + 5) Segm en t Cou p lin g (8a ,b). Compound
7 (1 equiv) was deprotected with TFA/DCM (1:1, v/v) for 30
min at ice temperature; then the solvent was removed in
vacuo. The residue was dissolved in the minimum amount of
DMF and neutralized with TEA under ice cooling. Then 2
equiv of 2a or 2b dissolved in a small amount of DMF was
added. The cooling was removed, and the solution was stirred
at room temperature. The reaction was monitored by TLC.
Then the volatiles were removed in vacuo, and the residual
oily material was purified by column chromatography using
chloroform/MEOH (9:1, v/v) containing 0.1% TEA.
Model 7125 injector equipped with
a 5-mL loop, and a
SpectraPhysics Spectra 100 UV/vis detector set at 216 nm. The
preparative column (25 mm × 100 mm cartridge with a Guard-
Pak insert from Waters Chromatography) was packed with
15-µm Delta-Pak C18. The mobile phase was mixed from 0.1%
(v/v) TFA in H2O and 0.08% (v/v) TFA in CH3CN at a 5.0 mL/
min flow rate.
Syn th esis. Nicotin oylp r olin e (Nic-P r o-OH, 1a ). In 100
mL of DCM, 1.95 g (11 mmol) of nicotinoyl chloride hydro-
chloride and 2.1 g (10 mmol) of Pro-tBu hydrochloride were
dissolved. Under ice cooling, 4.32 mL (21 mmol) of TEA was
added, and the cooling was removed. The solution was stirred
overnight and then extracted with 40 mL of 1 N HCl (2×), 40
mL of saturated NaHCO3 (2×), and brine. The organic layer
was dried and concentrated in vacuo. The residue (tert-butyl
ester) was diluted with an equivalent volume of TFA and
stirred overnight. After evaporation of the volatiles, the
residue was dissolved in water and neutralized with NaHCO3.
Then the aqueous phase was exhaustively extracted with
EtOAc. The combined organic layer was dried and evaporated
to a white, hygroscopic solid. After recrystallization from
EtOAc/ether 2.83 g (92% yield) of white solid was obtained.
1a : mp 88-92 °C; MS [M + H]+ m/ z 221. Anal. (C11H12N2O3)
C, H, N.
Nicotin oyla la n in e (Nic-Ala -OH, 1b): prepared similarly
to 1a with 89% yield after recrystallization from EtOAc/ether;
white, solid; mp 140-142 °C; MS [M + H]+ m/ z 195. Anal.
(C9H10N2O3) C, H, N.
Nicotin oylp r olin e P en ta flu or op h en yl Ester (Nic-P r o-
OP fp , 2a ). To the solution of 0.99 g (4.5 mmol) of 1a in 40
mL of EtOAc was added 0.91 g (4.9 mmol) of pentafluorophenol
Nic-P r o-DADLE-OCh o (8a ): white solid; mp 175-179 °C;
Rf ) 0.41; MS [M + H]+ m/ z 1140. Anal. (C67H93N7O9‚H2O)
C, H, N.
Nic-Ala -DADLE-OCh o (8b): white solid; mp >200 °C; Rf
) 0.36; MS [M + H]+ m/ z 1114. Anal. (C65H91N7O9‚H2O) C,
H, N.
Boc-D-leu cin e Ad a m a n t ylet h yl E st er (Boc-D-Leu -
OAd a , 11). 11 was Prepared similarly to 3 with 92% yield.
An analytical sample was obtained after column chromato-
graphic purification: oily material; Rf ) 0.49; MS [M + Na]+
m/ z 415. Anal. (C23H39NO4‚0.25 H2O) C, H, N.
Solid P h a se P ep tid e Syn th esis (12a ,b). Fmoc-Phe resin
(3.0 g, 2.34 mmol) was placed in a 250-mL reaction vessel, and
the stepwise SPPS was carried out as follows: (1) deprotection
with a 9-min cycle of piperidine/NMP (1:1, v/v), (2) washing,
NMP (3 × 2 min), MeOH (3 × 2 min), and NMP (3 × 2 min),
(3) coupling, Fmoc-amino acid (2.5 equiv), DCC (2.5 equiv),
and HOBt (2.5 equiv) in NMP, and (4) washing, NMP (3 × 2
min), MeOH (3 × 2 min), and NMP (3 × 2 min). The coupling
time was set for 4-10 h, and usually double coupling was
applied. The completeness of the reaction was monitored by
the Kaiser test.32 After the final coupling the resin was
washed as before and dried in vacuo. Cleavage was effected
by TFA/H2O (95:5, v/v) for 90 min. The solution was concen-
trated; the residue was dissolved in glacial acetic acid and
freeze-dried. The crude peptide was shown to be about 90%
pure by analytical RP-HPLC. Compound 12 was used without
purification. Analytical samples were obtained after prepara-
tive RP-HPLC purification using gradient elution.
under ice cooling followed by 1.00 g (4.9 mmol) of DCC.
A
precipitate formed almost immediately. After 30 min, the
cooling was removed, and the solution was stirred for 2 h. The
precipitate (urea) was filtered off and discarded. The filtrate
was washed with 20 mL of 0.1 N HCl (2×), 20 mL of saturated
NaHCO3 (2×), and 20 mL of brine (2×). The organic layer
was dried and evaporated. The obtained solid was recrystal-
lized from EtOAc/ether resulting in 1.46 g (84% yield) of white
solid: mp 65-68 °C; Rf ) 0.88; MS [M + H]+ m/ z 387. Anal.
(C17H11N2O3F5) C, H, N,
Nicotin oyla la n in e p en ta flu or op h en yl ester (Nic-Ala -
OP fp , 2b): prepared as 2a with 89% yield; recrystallized from
EtOAc/hexane; white solid; mp 110 °C; Rf ) 0.79; MS [M +
H]+ m/ z 361. Anal. (C15H9N2O3F5) C, H, N.
Boc-D-leu cin e Ch olester yl Ester (Boc-D-Leu -OCh o, 3).
The solution of 2.31 g (10.0 mmol) of Boc-D-leucine, 5.72 g (11
mmol) of PyBOP, and 4.4 mL (25 mmol) of DIEA in 200 mL of
DCM was stirred under ice for 1 h; then 3.5 g (9 mmol) of
cholesterol was added. The solution was stirred at room
temperature overnight and then extracted with 50 mL of 0.1
N HCl (2×) and 50 mL of saturated NaHCO3. The organic
layer was dried and evaporated to an oil that slowly crystal-
lized on standing. The crude product was recrystallized from
EtOAc/hexane (89% yield) and used without further purifica-
tion. An analytical sample was obtained after column chro-
matographic purification using chloroform containing 0.05%
of TEA: white solid; mp 107-109 °C; Rf ) 0.58; MS [M + Na]+
m/ z 623. Anal. (C38H65NO4‚H2O) C, H, N.
Gen er a l P r oced u r e for P ep tid e Ch a in Elon ga tion
Usin g Am in o Acid Active Ester (4-7). The Boc-protected
compound (3-6) was deprotected with TFA/DCM (1:1, v/v) for
30 min at ice temperature; then the volatiles were removed
by exhaustive evaporation. The residue was dissolved in the
minimum amount of DCM and/or DMF and neutralized with
Nic-P r o-Tyr -D-Ala -Gly-P h e-OH tr iflu or oa ceta te (12a ):
no definite melting point; MS [M + H]+ m/ z 659. Anal.
(C36H38N6O10F3‚0.25TFA‚0.75H2O) C, H, N.
Nic-Ala -Tyr -D-Ala -Gly-P h e-OH tr iflu or oa ceta te (12b):
no definite melting point; MS [M + H]+ m/ z 633. Anal.
(C34H36N6O10F3‚0.5 TFA‚H2O) C, N, H.
Meth od B: (6 + 1) Segm en t Cou p lin g (13a ,b). In a
solvent mixture of DMF and dioxane, 1 equiv of unpurified
12a or 12b, 1.1. equiv of PyBOP, and 3.5 equiv of DIEA were
dissolved. The solution was stirred at ice temperature for ca.
1 h. In the meantime, in a separate flask 11 was deprotected
in a customary manner. After removing the solvents, the
residue (TFA salt of Leu ester) was dissolved in a minimum
amount of DMF and neutralized with DIEA. This solution was
added to the coupling milieu. The reaction mixture was stirred
at room temperature overnight and then evaporated to an oily
residue and purified similarly to 8a ,b.
Nic-P r o-DADLE-OAd a (13a ): white solid; mp 155-165
°C dec; Rf ) 0.56; MS [M + H]+ m/ z 934. Anal. (C52H67N7-
O9‚1.75 H2O) C, H, N.

CDS of a Leu-enkephalin Analogue
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24 4781
Nic-Ala -DADLE-OAd a (13b): white solid; mp 220-223 °C;
Rf ) 0.53; MS [M + H]+ m/ z 908. Anal. (C50H65N7O9‚1.5 H2O)
C, H, N.
mobile phase was mixed from 0.1% (v/v) TFA in water and
0.08% (v/v) TFA in acetonitrile, and gradient elution was done
from 5% to 95% organic modifier that changed in a linear
profile at a rate of 1%/min. HPLC calibration curves for
determining concentrations were obtained by adding known
amounts of peptide into aliquots of brain homogenate trans-
ferred into ice cold 1 M acetic acid solution and analyzing the
supernatant after centrifugation. Concetration-time profiles
were analyzed by exponential fitting, assuming a pseudo-first-
order degradation. Half-lives (t1/2) were calculated from the
rate constants (k) as 0.693/k. Representative samples were
desalted on a reversed phase minicolumn (1-mL Supelclean
LC-18, Supelco, Bellefonte, PA) and analyzed by electrospray
ionization mass spectrometry (Vestec/PerSeptive Biosystems,
Model 200 ES instrument) to ascertain the release of DADLE
from 16.
Op ioid Recep tor Bin d in g. Opioid receptor assays used
rat brain membrane preparations and measured binding
affinities versus known potent ligands with standardized
rapid-filtration techniques. Adult male Sprague-Dawley rats
(200-220 g) were decapitated, the brain was removed and
chilled on ice, and then tissue was suspended in about 30 mL
of cold 50 mM Tris HCl buffer containing 100 mM NaCl and
adjusted to pH 7.4 with HCl. The suspension was homog-
enized in a motor-driven Potter-Evjen tissue grinder, and the
homogenate was centrifuged at 2000 rpm for 15 min at 5 °C.
The pellet was resuspended in sodium-free Tris HCl buffer,
washed twice in the same media before the suspension was
aliquoted, and stored frozen at -50 °C. Receptor binding
affinity was determined from competitive displacement with
potent opioid agonists [3H]diprenorpine (µ and δ) or [3H]DAGO
(µ-selective). The brain preparation was diluted with Tris HCl
buffer to a final concentration of 0.5% and preincubated to
remove endogenous opioids. The brain receptor preparation
(100 µL aliquots) was then incubated with labeled opioid
agonist (0.5-1.0 nM) in a total volume of 1 mL of 50 mM Tris
HCl, pH 7.4, containing 30 µM bestatin, 0.6 µM tiorphan, 10
µM captopril, and 2 µM Leu-Leu for 3 h at 25 °C.33 The
samples were filtered with a Brandel cell harvester through
GF/B Whatman glass-fiber filter strips pretreated with 0.1%
poly(ethylenimine) solution. Three washes with ice cold saline
were included before assaying bound radioactivity by liquid
scintillation spectrophotometry. Nonspecific binding was
determined in the presence of excess naloxone (1 µM). The
IC50 values were calculated from standard curves of %Rx versus
concentration of the added peptide:
Gen er a l P r oced u r e for P r ep a r a tion of T+-S-DADLE-
OLp f (9a ,b a n d 14a ,b). Compound Nic-S-DADLE-OLpf (1
equiv) was dissolved in chloroform/MeOH, and ca. 20 equiv of
MeI was added. The solution was stirred at room temperature
overnight. Ether was added, and the obtained yellow, pasty
precipitate was filtered off and recrystallized (2×) from
chloroform/MeOH (8:2, v/v) and EtOAc. The product was
obtained quantitatively.
Tr igon ellyl-P r o-DADLE-OCh o iod id e (9a ): yellow solid;
mp 195-197 °C dec; Rf ) 0; MS Q+ (quaternary pyridinium
ion) m/ z 1154; UVmax 264 nm. Anal. (C68H96N7O9I‚1.5 H2O‚0.5
MeI) C, H, N, I.
Tr igon ellyl-Ala -DADLE-OCh o iod id e (9b): yellow solid;
mp 164-166 °C dec; Rf ) 0; MS Q+ m/ z 1128; UVmax 264 nm.
Anal. (C66H94N7O9I‚1.5 H2O‚0.5 MeI) C, H, N, I.
Tr igon ellyl-P r o-DADLE-OAd a iod id e (14a ): yellow solid;
mp 145-149 °C (dec); Rf ) 0; MS Q+ m/ z 948; UVmax 264 nm.
Anal. (C53H70N7O9I‚1.75 H2O) C, H, N, I.
Tr igon ellyl-Ala-DADLE-OAda iodide (14b): yellow solid;
mp 173-173 °C (dec); Rf ) 0; MS Q+ m/ z 922; UVmax 264 nm.
Anal. (C51H68N7O9I‚1.5 H2O) C, H, N, I.
Gen er a l P r oced u r e for Sod iu m Dith ion ite Red u ction
(10a ,b a n d 15a ,b). The pyridinium salts (1 equiv), 9a ,b, and
14a ,b were dissolved in a mixture of ethanol/water (ca. 1:1,
v/v). The solution was kept under ice cooling and degassed
with continuous nitrogen bubbling for ca. 20 min before the
mixture of Na2S2O4 (4-5 equiv) and Na2CO3 (6-7 equiv) was
added. The pH of the reaction mixture was kept around 7.0-
7.2. The cooling and the nitrogen bubbling continued during
the reduction (3-4 h) monitored by TLC and UV spectroscopy.
Then the reaction mixture was diluted with ice cold, degassed
chloroform and extracted with ice cold, degassed water. The
layers were separated under a nitrogen atmosphere. The
organic layer was dried and carefully evaporated to a pale
yellow solid that was recrystallized from degassed chloroform/
MEOH (8:2, v/v) and ether in an inert atmosphere. The yield
was around 60%.
1,4-Dih yd r ot r igon ellyl-P r o-DADLE -OCh o (10a ): Rf )
0.53; MS [M + Na]+ m/ z 1177; UVmax 360 nm.
1,4-Dih yd r otr igon ellyl-Ala -DADLE-OCh o (10b): Rf
0.48; MS [M + Na]+ m/ z 1151; UVmax 360 nm.
1,4-Dih yd r otr igon ellyl-P r o-DADLE-OAd a (15a ): Rf
0.34; MS [M + Na]+ m/ z 971; UVmax 360 nm.
)
)
)
1,4-Dih yd r otr igon ellyl-Ala -DADLE-OAd a (15b): Rf
%Rx ) 100[(Cpmx - CpmN)/(CpmL - CpmN)]
where CpmL is the mean value with only the radiolabeled
ligand and %RL ) 0 and CpmN is the mean value with
ligand and excess naloxone and %RN ) 100.
0.34; MS [M + Na]+ m/ z 945; UVmax 360 nm.
Tr igon ellyl-P r o-DADLE-OH tr iflu or oacetate (16a): pre-
pared by SPPS similarly to 12; MS Q+ m/ z 786. Anal.
(C43H52N7O11F3‚2 H2O) C, H, N.
Tr igon ellyl-Ala-DADLE-OH tr iflu or oacetate (16b): pre-
pared by SPPS similarly to 12b; MS Q+ m/ z 760. Anal.
(C41H50N7O11F3‚1.5 H2O) C, H, N.
P h a r m a cology. CDSs were evaluated for CNS-mediated
analgesia in Sprague-Dawley rats (250-350 g of body weight).
The latency of the tail-flick response to radiant heat (focused
in the middle of the tail) was measured by using a Tail-Flick
Analgesia Meter 0570-001L (Columbus Instruments Interna-
tional Corp., Columbus, OH). Base-line measurements (2-3)
were collected prior to drug administration, and tail-flick
latency was measured at various times up to 6 h following drug
administration. Groups of 5-10 animals were treated with
vehicle control, the solution of the unmodified peptide, and its
CDSs, respectively. The peptide and CDSs were administered
in 4.2 µmol/kg of body weight doses. For the study involving
the opioid antagonist naloxone and its quaternary methyl
analogue, 15 rats (divided into three groups of five) were
injected with a dose of 4.2 µmol/kg of body weight CDS, iv, in
0.1 mL of vehicle solution. After 30 min, animals in two groups
were injected subcutaneously with naloxone and naloxone
methiodide at a dose of 1 mg/kg of body weight, respectively,
and the tail-flick latency was monitored for an additional 90
min in 30-min intervals. Data generated from the studies
comparing treatment groups were subjected to analysis of
variance (ANOVA) followed by Dunnett’s procedure to detect
significant differences between groups with p > 0.05 selected
as the level of significance.
In Vitr o Sta bility a n d Meta bolism Stu d ies. Approxi-
mately 250 nmol of CDS or its oxidized form (T+), dissolved in
DMSO, or 30 nmol of 16 was added to 1 mL of rat brain
homogenate (20%, w/w, in pH 7.4 Tris buffer), respectively,
and the mixture was incubated at 37 °C in a temperature-
controlled, shaking water bath. Aliquots (100 µL) were
removed after 5, 15, 30, 45, 60, and 90 min of incubation,
respectively, and transferred to 1.5-mL plastic centrifuge tubes
containing 200 µL of ice cold 1 M aqueous acetic acid. After
centrifugation at 12500g for 15 min, the supernatant was
removed and analyzed by microbore HPLC. Analyses were
done on a system consisting of a ThermoSeparation/Spectra-
Physics (Fremont, CA) Spectra Series P200 binary gradient
solvent delivery system, a Rheodyne (Cotati, CA) Model 7125
injector valve equipped with a 5-µL sample loop, a Spectroflow
757 variable wavelength UV/vis detector (Kratos Analytical,
Manchester, U.K.) operated at 216 nm, and a Hewlett-Packard
Model HP 3395 computing integrator (Palo Alto, CA). A 30
cm × 1.0 mm i.d. Supelcosil LC-18 (5 µm) reversed phase
column (Supelco, Bellefonte, PA) was used at a flow rate of 50
µL/min that was maintained by using a dynamic split (before
the injection valve) via a balance column (25 mm × 4.6 mm
i.d) connected in parallel with the microbore column. The
F or m u la tion of th e CDS. The freshly prepared com-
pounds 10a ,b and 15a ,b were dissolved in the mixture (9:1,

4782 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 24
Prokai-Tatrai et al.
v/v) of (2-hydroxypropyl)-â-cyclodextrin (HPâCD)34 (50%, w/v,
aqueous solution) and DMSO35 for the tail-flick latency
measurements. We have also studied the solubility of our
peptide CDSs in HPâCD for optimizing their formulation. The
cyclodextrin inclusion complexes were prepared by equilibrat-
ing an excess of CDSs with a 50% (w/v) aqueous solution of
HPâCD by the following way: To the degassed solution of
HPâCD was added a known amount of CDS. The suspension
was sonicated for 30 min under ice cooling and then filtered,
and the filtrate was lyophilized. The CDS content of the
complexes was analyzed. The degree of complexation was
around 20 mg/g.
(16) Bodor, N. Drug Targeting and Retrometabolic Drug Design
Approaches. Adv. Drug Delivery Rev. 1994, 14, 157-166.
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M.; Kaminski, J .; Bodor, N. Reactivity of biologically important
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1,4-dihydronicotinamide and its corresponding pyridinium salt.
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Kaminski, J . Reactivity of biologically important reduced pyr-
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proline cleaving enzyme and post-proline dipeptidyl aminopep-
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W. Structure-activity relationship of LHRH antagonists: Incor-
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In Peptides: Chemistry, Structure and Biology; Proceedings of
the Thirteenth American Peptide Symposium; Hodges, R. S.,
Smith, J . A., Eds.; ESCOM: Leiden, 1994, pp 565-567.
(23) Tsuzuki, I.; Hama, T.; Hibi, T.; Konishi, R.; Futaki, S.; Kitagawa,
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Ack n ow led gm en t. This project has been supported
by the National Institute for Drug Abuse (Grant No.
RO1 DA09268) and by a National Institutes of Health
Training Grant (T32 NS07333). Appreciation is ex-
pressed to Drs. W. M. Wu, E. Brunt-Chikhale, and G.
Hochhaus as well as to Mr. H. S. Kim for their
invaluable help. The technical assistance of Dr. Z.
Szeiler is acknowledged.
Su p p or tin g In for m a tion Ava ila ble: Electrospray ioniza-
tion mass spectrometric analysis showing the release of
DADLE in rat brain homogenate in vitro (1 page). Ordering
information is given on any current masthead page.
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